Enteropathogenic Escherichia coli (EPEC) are a leading cause of diarrhea among infants in developing countries. EPEC attach intimately to the membrane of target cells, efface epithelial microvilli, and induce profound changes in the underlying cytoskeleton. TnphoA mutagenesis has revealed a 13.8 kilobase chromosomal region required for these effects. Mutations in the eae gene, which has homology with the inv gene of Yersinia pseudotuberculosis, and at least two genes downstream of eae interfere with the ability to attach intimately to epithelial cells and disrupt microvilli and cellular actin. These properties are restored upon reintroduction of these genes in trans. The central hypothesis of this proposal is that the eae gene cluster is responsible for the close attachment of EPEC to the intestinal epithelial cell membrane with subsequent effects on the cytoskeleton, and that the product of the eae gene is the actual adhesin involved. To test this hypothesis, binding studies will be performed to evaluate the role of the Eae product in attachment to tissue culture cells. The receptors recognized by Eae will be sought, and the role of the Eae product in epithelial cell calcium metabolism and protein kinase C activation will be investigated. The eae gene cluster will be mapped to determine the precise location and nucleotide sequences of the genes required for attaching and effacing activity. The transcriptional organization of the region will be investigated by Northern hybridization and primer extension analyses. Protein products will be identified by in vitro transcription-translation and minicell analyses and antisera against these products will be raised. The interactions among genes of the eae cluster will be investigated with particular attention to possible post-translational modification of the Eae product and protein-protein interactions. It is expected that these studies will shed light on the pathogenesis of EPEC infection and of bacterial-cellular interactions in general.